1. Field of the Invention
This invention relates in general to an animal model that is useful for developing therapeutic drugs of a disease. More specifically, the present invention relates to the creation of a transgenic animal having an exogenous gene construct coding a part of .beta.-amyloid precursor protein (hereinafter called APP) in its genome. The exogenous gene construct is designed to overexpress in various types of the cells.
2. Description of the Prior Art
Recent development of genetic engineering has made it possible to create embryos (so-called transformed embryos) into which a gene construct is integrated by microinjection of an exogenous gene construct (DNA) into the nuclei of 1-cell stage embryos or by infection of preimplantation embryos with retroviral vector DNA (Gordon et al., Proc. Natl. Acad. Sci., U.S.A., 77: 7380 (1980); Jaenisch et al., Cell, 32: 209 (1983). The resulting embryos can further develop to full term after the transfer into the oviducts/uteri of recipient foster mothers. Some of the resulting adult animals have the exogenous DNA integrated into their genome and to express the DNA in its tissues. These "transformed" animals are generally called transgenic animals (Gordon et al., Science, 214: 1244 (1981). The integrated exogenous DNA is called a transgene, generally consisting of a promoter and a target gene (encoding a protein that is desired to be expressed) and other regulator sequences. Expression of the transgene can occur even before maturation; for example, in some cases, the expression occurs in the cleavage stage of an embryo. As a result of the expression, a protein encoded by the transgene is produced. If this protein plays a crucial role on the morphogenetic pathway of individuals, some phenotypic alteration may occur at a certain stage of development. To provide phenotypic alteration in transgenic animals, two approaches are possible; 1) overexpression of a target protein in a targeted tissue(s) and 2) suppression of endogenous target gene expression by anti-sense gene technology (see. e.g., Katsuki et al., Science, 241: 593 (1988)). These are based upon an usage of tissue-specific or ubiquitous promoter and/or enhancer, both of which should be placed upstream of the target gene.
There are many reports that demonstrate that transgenic animals exhibit alteration of their original phenotypes due to expression of a transgene. These are mentioned in detail in the reviews by Palmiter et al. Annu. Rev. of Genet. 20: 465 (1986) and Cordon, Int. Rev. of Cytobiol. 115: 171 (1989), among others. These transgenic animals can be utilized in the fields of 1) analysis of gene expression in vivo during embryogenesis, 2) gene therapy for overcoming hereditary genetical diseases, and, 3) as a test system for pharmaceutical compositions. Transformation of an embryo with DNA can be achieved by giving the DNA exogenously; the added DNA then will be integrated into a part of DNA sequence in the chromosomes of the host embryos. Several techniques are known for introducing the exogenous DNA into mammalian embryos. For example, DNA may be introduced via a micropipett (so-called microinjection method) into the pronuclei of one-cell stage embryos (Gordon et al., 1980, above).
By using microinjection method, mammalian embryos into which DNA is injected can develop to full term after transfer to the oviducts or uteri of pseudopregnant female recipients. The progeny can be analyzed later by PCR (polymerase chain reaction) and/or Southern blot method to determine whether or not they have the injected DNA in their chromosomes. If the presence of the exogenous DNA is confirmed, the transgenic animals can next analyzed for gene expression by Northern blot hybridization or immuno-histochemical methods. In this way, it is possible to introduce a certain human hereditary disease-like character into an animal.
Alzheimer's disease (hereinafter called AD) is considered, as mentioned later in detail, to be caused by overexpression of APP (Terry et al., Ann Neurol, 14: 496 (1983)). In the brains of patients with AD, there are observed neurofibrillary tangles (hereinafter called NFT), paired helical filaments (hereinafter called PHF), a neuritic plaque (or a senile plaque) and deposition of cerebral amyloid which are peculiar to AD; the latter two structures are derived from APP. Moreover, mutations in the APP gene have been recently found in familial Alzheimer's disease (hereinafter called FAD) and in hereditary cerebral amyloid angiopathy. In addition, it has been reported that amyloid plaque core protein (hereinafter called APCP), one of major components of cerebral amyloid, or D-amyloid core protein (later renamed D-protein or D/A4 protein; hereinafter called fl/A4 protein) is toxic to neurons (Yankner et al., Science, 245: 417 (1989)). From these data, it is considered that the most essential approach to elucidate the pathogenesis of AD is to analyze how fl/A4 protein is metabolized from APP and finally deposited in a brain.
As mentioned previously, there are two distinct morphological and pathological changes associated with AD, namely, formation of PHF and deposition of cerebral amyloid. PHF appears more often in other neuronal diseases than AD; whereas, both the neuritic plaque which is an amyloid deposit generated in an intercellular space of neurons and amyloid deposited in the periphery of cerebral blood vessels are considered to be specific for AD. Interestingly, the neuritic plaques are also observed in the brain of aged patients with Down's syndrome (AD is also occurring). Amyloid proteins, a major component of the neuritic plaque, were partially purified and found to consist of mainly .beta./A4 protein with about 4.2 kD comprising 39 to 42 amino acids (Glenner et al., BBRC, 120: 1131 (1984)). The amino acid sequence of .beta./A4 protein was determined (Glenner et al., 1984; Masters et al., Proc. Natl. Acad. Sci. U.S.A., 82: 4245 (1985) and proved different from the proteins previously reported so far.
A cDNA encoding APP, a relatively large protein precursor including .beta./A4 protein part, was recently isolated from the cDNA library of human embryonic cerebral tissues. Analysis of the DNA sequence of human APP cDNA revealed that human APP consists of 695 amino acids (hereinafter called A695), and .beta./A4 protein corresponds to the amino acid positions at 597 to 695 (Kang at al, Nature, 325: 733 (1987)). Furthermore, beside A695, successful isolation of other two cDNAs for larger APPs (hereinafter called A751 and A770, respectively) was reported (Kitaguchi at al., Nature, 331: 530 (1988)). A751 is a protein consisting A695 with a 56-amino acid insert. The 56-amino acid insert shows a high homology to serine protease inhibitor (hereinafter called KPI) of Kunitz family (Kitaguchi et al., 1988). A770 is a protein in which a 19 amino acid sequence highly homologous to MRC OX-2 antigen is inserted immediately after the 56 amino acid insert in A751 (Kitaguchi et al., 1988). These A751 and A770 proteins are abundant in many tissues. These three types of proteins are known to be generated by alternative splicing from the same APP gene (Kitaguchi at al., 1988; Ponte et al., Nature, 331: 525 (1988); Tanz et al., Nature, 331: 528 (1988). They are thought to be involved in cerebral amyloid deposition, because each has the .beta./A4 protein portion located on the C-terminal 99 amino acid fragment of APP (the N-terminal 28 amino acid part of this fragment is exposed outside the cell membrane, whereas a domain of .beta./A4 protein at its C-terminal side, comprising of 11-14 amino acids, exists inside of the cell membrane).
Immunohistochemical studies in the brains of patients with AD by using various antibodies raised against peptides corresponding to the several sites of APP have revealed that neuritic plaques can be stained by these antibodies (Wong et al., Proc. Natl. Acad. Sci., U.S.A., 82: 8729 (1985); Allsop et al., Neurosci. Lett., 68: 252 (1986); Shoji et al., Brain Res., 512: 164, (1990); Shoji et al., Am. J. Pathol., 137: 1027 (1990); Shoji et al., Brain Res., 530: 113 (1990). Therefore, amyloid proteins composing neuritic plaques in the AD patients can be easily recognized by these antibodies. By using these antibodies, one can trace the localization of APP and its metabolized derivatives in a brain of a transgenic animal overexpressing a human APP gene.
As APP is widely expressed in many tissues and is also evolutionally con served (there is a 97% homology at the amino acid level between mouse and human), it is postulated to play an important role an cell-cell interaction and/or neuronal cell differentiation (Shivers at al., EMBO. J., 7: 1365 (1988). Its precise role, however, is still unclear. Recently, it drew an attention that .beta./A4 protein at lower concentrations serve s as a growth stimulating factor for hippocampal matured neuronal calls, but it is neurotoxic at higher concentrations (Yankner et al., 1989). Interestingly, it was shown that the portion corresponding to the N-terminal 25th to 35th amino acid of .beta.A4 protein is essential for both growth stimulating and inhibitory activities, and is homologous to the tachyquinin-type peptides (Yankner et al., 1989). More interestingly, when the purified .beta./A4 protein was injected into a cerebral cortex and a hippocampus of rats, a neuronal cell loss was induced as well as production of abnormally phosphorylated tau protein, a major constituent of PHF (Kowall et al., Proc. Natl. Acad. Sci., U.S.A., 88: 7247 (1991). These data suggest a close relationship between accumulation of .beta./A4 protein and PHF production. As another aspects of the role of APP, it has been reported that the C-terminal region of APP can be phosphorylated by protein kinase C and Ca.sup.2+ /calmodulin-dependent protein kinase II (Gandy et al., Proc. Natl. Acad. Sci., U.S.A., 85: 6218 (1988), and that G.sub.o protein, a major GTP-binding protein present beneath the cell membrane, can interact with APP (Nishimoto et al., Nature, 362: 75 (1993). These data suggest that APP is involved in signal transduction.
The APP gene is located on the long arm of the 21st chromosome in human (Goldgaber et al., Science, 235: 877 (1987)). Recently, in FAD (AD frequently occurs in people earlier than 65 years old), a mutation (from Val to Ile) was found at the amino acid position of 642 in human APP (based on the data of Kang et al., 1987; hereinafter, DNA and amino acid sequences of APP are based on the data of Kang et al., 1987) (Goate et al., Nature, 349: 704 (1991); Naruse et al., Lancet, 337: 978 (1991); Yoshioka et al., BBRC, 178: 1141 (1991); Hardy at al., Lancet, 357: 1342 (1991). Furthermore, other mutations (Val to Phe and Val to Gly) at 642 in APP have been found (Murrell J. et al., Science, 254: 97 (1991). These data suggest that mutation at Val.sup.642 of APP would play an important role on the pathogenesis of FAD. In the case of Dutch-type AD which is frequently associated with hereditary cerebral hemorrhage, a mutation (Glu.sup.618 to Gln.sup.618) was found within the .beta./A4 protein part (Levy et al., Science, 248: 1124 (1990). Furthermore, two mutations (Lys.sup.595 to Asn.sup.595 and Met.sup.596 to Leu.sup.596) at the N-terminus of .beta./A4 protein were recently found in AD patients from a certain Swedish family (Mullan at al., Nature, Genet, 1: 345 (1992). This type of AD is called Swedish-type AD.
As described above, the molecular biological analysis of APP has been developed, but any effective information is not available yet as to why amyloid is accumulated and deposited in the brain of patients with AD, and how a neuronal cell is degenerated as a result of accumulation of .beta./A4 protein.
The present most exciting problem is that what kind of metabolic pathway of APP is profoundly involved in cerebral amyloid deposition. This matter is now being investigated extensively. For example, a membrane-bound C-terminal fraction of APP, 9 KD, could be extracted from human embryonic kidney-derived cell line 293 which had been transfected with expression vector DNA for APP cDNA, and the amino acid sequence of the N-terminus of the 9-kDa peptide was determined. As a result, APP was cleaved at the 16th Lys from the N-terminus of .beta./A4 protein (Esch at al., Science, 248: 1122 (1990). However, for deposition of cerebral amyloid, it requires that APP should be cleaved at both N-terminus and C-terminus of .beta./A4 protein and then aggregated. Therefore, insoluble .beta./A4 protein is not produced by the metabolic pathway of APP provided by Esch et al. (1990). Now, involvement of various metabolic systems and their defects would be considered as key factors for amyloid formation, but no clear answers have yet been obtained.
At present, it is considered that there are two pathways for APP processing; namely, 1) so-called secretary pathway, in which APP is processed into the secreted derivatives with molecular weights more than 100 KD ending at the 15th amino acid of .beta./A4 protein, and 2) so-called endosomal/lysosomal pathway, in which various APP peptides that are different in size but including a full length .beta./A4 protein portion are generated (Golde at al., Science, 235: 728 (1992).
Consequently, it has not yet been resolved how these two possible APP metabolic pathways are influenced by the mutations in the APP gene found in FAD, Dutch-type and Swedish-type AD. Probably, these APP mutants may allow the APP processing pathway to be entered into the endosomal/lysosomal pathway, not into the secretary pathway. In this connection, a transgenic animal system, in which overexpression of APP mutants are forced to be driven, will provide an useful tool for elucidating the mechanism underlying APP processing.
Recently, several reports have appeared relating to transgenic mice in which amyloid deposition was observed in their brains by overexpression of the full length or one part of human APP cDNA (Kawabata et al., Nature, 345: 476 (1991); Quon et al., Nature, 352: 239 (1991); Wirak at al. Science, 253: 323 (1991). However, the report by Kawabata at al. (1991) was later found to be not reproducible, and the paper was recently withdrawn (Nature, 356: 265, (1992). Furthermore, the report by Wirak at al. (1991) was later found to be misleading because the phenotypic change to AD-like character in APP-overexpressing transgenic mice was not caused by transgene expression (Science, 28, February, 1992). In addition, several published patent applications have reported that mouse transgenic model for AD are established (WO93/14200, W093/02189, W092/13069, W092/06187, W091/19810, EP451700 and WO 89/06689). However, in each of these applications gene construction was only mentioned and/or only mentioned with indirect proofs only that the production of APP (not .beta./A4 protein) was observed. Furthermore, neither a neuronal cell loss nor formation of a neuritic plaque was mentioned in any report. Consequently, specific and distinct animal models for AD produced by genetic engineering technology are considered not yet to be established.
An important need therefore persists for an animal model of AD that exhibits the physiological and biochemical characteristics of this disease as described in human patients. This need has now been satisfied with the transgenic animal model of AD described below.